本文選題：E-cadherin + 樹突狀細(xì)胞�。� 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：研究背景及目的：肺癌是我國最常見的惡性腫瘤之一,據(jù)腫瘤登記中心報道,2010年,中國新發(fā)肺癌病例60.59萬,居惡性腫瘤首位,同期我國肺癌死亡人數(shù)為48.66萬,占惡性腫瘤死因的24.87%。目前針對肺癌的治療方式主要有手術(shù),放療,化療及靶向治療等治療方式,但是目前還沒有一種傳統(tǒng)的治療方式可以根治肺癌。隨著治療方式的不斷發(fā)展和更新,免疫治療及對肺癌免疫微環(huán)境的發(fā)病機制的研究已進(jìn)入新的時代,這為肺癌的個體化治療提供了新的治療思路和徹底治愈肺癌帶來了希望。由于肺癌這類實體腫瘤,與惡性黑色素瘤不同,不具備免疫原性,在腫瘤微環(huán)境中不易被T細(xì)胞發(fā)現(xiàn)及結(jié)合,同時自身能促進(jìn)調(diào)節(jié)T細(xì)胞(Treg)分化及其相應(yīng)細(xì)胞因子的分泌,從而抑制自身抗腫瘤T細(xì)胞反應(yīng)。目前肺癌免疫治療針對增強抗腫瘤T細(xì)胞反應(yīng)的主要方法是用單克隆抗體(如anti-PD-1和anti-CTLA-4。)阻滯抑制T細(xì)胞激活通路的檢查點。 但最近共刺激單克隆抗體anti-CD40為肺癌免疫治療提供了又一新的免疫治療思路,而且目前已經(jīng)在胰腺癌,彌漫大B細(xì)胞淋巴瘤一期臨床研究中展現(xiàn)出良好的抗腫瘤效果。但是anti-CD40刺激機體后又具備細(xì)胞因子釋放綜合征,血栓等不良反應(yīng),從而限制了其廣泛的應(yīng)用。我們知道,Anti-CD40具備激活抗原提呈細(xì)胞(APC)誘導(dǎo)炎癥反應(yīng)及腫瘤微環(huán)境中具備良好抗腫瘤的雙重作用。為了彌補anti-CD40的不足,研究anti-CD40介導(dǎo)的炎癥反應(yīng)中炎性樹突狀細(xì)胞抗腫瘤的機制研究將為anti-CD40單克隆抗體更好的應(yīng)用于肺癌樹突狀細(xì)胞免疫治療領(lǐng)域提供良好的理論依據(jù)。同時,以樹突狀細(xì)胞(DCs)為基礎(chǔ)的肺癌免疫治療手段已經(jīng)成為新的研究熱點。但不同DCs亞群生物學(xué)功能及其調(diào)控機制尚未明確,這將成為導(dǎo)致其臨床應(yīng)用療效不佳的重要原因。而最新研究發(fā)現(xiàn)Anti-CD40誘導(dǎo)的炎癥反應(yīng)中,一類新型的炎癥樹突狀細(xì)胞亞型E-cadherin陽性樹突狀細(xì)胞被Fiona Powrie教授發(fā)現(xiàn)。但是此類樹突狀細(xì)胞在肺癌免疫微環(huán)境中的作用及機制未知,本實驗主要是探索該新型炎性E-cadherin陽性樹突狀細(xì)胞在lewis市癌模型抗腫瘤T細(xì)胞免疫中的作用及機制。 材料與方法：1.利用FGK45細(xì)胞上清制備鼠anti-CD40抗體。然后利用rag1KO小鼠及anti-CD40抗體制備anti-CD40炎癥動物模型。2.分別取Lewis肺癌模型,C57/BL6小鼠,Rag1KO小鼠及其anti-CD40炎癥動物模型小鼠的肺組織,提取此4種免疫狀態(tài)的肺組織及其單個細(xì)胞,分別利用共聚焦技術(shù)和流式檢測技術(shù)檢測炎性E-cadherin陽性樹突狀細(xì)胞(DC)的分布及表型。3.提取anti-CD40炎癥動物模型脾臟來源的單個細(xì)胞,流式分選出E-cadherin+DC,并攜帶CEA421-435抗原肽與初始(naive) CD4+T細(xì)胞共培養(yǎng),利用胞內(nèi),胞外細(xì)胞因子檢測法分析anti-CD40誘導(dǎo)的炎性E-cadherin+DC在腫瘤微環(huán)境下激活初始CD4+T細(xì)胞向效應(yīng)(effector) T細(xì)胞(Thl, Th2及Th17)及調(diào)節(jié)T細(xì)胞(Treg)分化情況。4.將E-cadherin+DC攜帶CEA526-533抗原肽與初始(naive) CD8+T細(xì)胞共培養(yǎng),利用ELISA技術(shù)分析其刺激效應(yīng)CD8+T細(xì)胞產(chǎn)生IFN-γ的水平。5.將anti-CD40誘導(dǎo)的E-cadherin+DCs負(fù)載CEA526-533抗原肽通過尾靜脈注入lewis肺癌原位模型,檢測其體內(nèi)CEA tetramer+(四聚體)CD8+T細(xì)胞動態(tài)變化及CD103+CD8+T細(xì)胞變化。同時在第28天取肺癌及肺組織檢測其微環(huán)境CD4+T細(xì)胞亞群變化。 結(jié)果：1.成功制備高純度anti-CD40抗體和anti-CD40炎癥動物模型。2.E-cadherin+DC主要大量聚集在anti-CD40炎癥動物模型的肺部,而在lewis市癌模型,正常C57/BL6小鼠及Rag1KO小鼠肺部未見明顯聚集。肺部anti-CD40誘導(dǎo)的炎性E-cadherin+DC類似脾臟炎性E-cadherin+DC表型,都是E-cadherin+CD11chighCD11b+CD103-CD4-.3. E-cadherin+DC攜帶CEA抗原肽在體外促進(jìn)了初始CD4+T細(xì)胞向Thl, Th17細(xì)胞分化,抑制了Th2和Treg細(xì)胞的分化。4.較E-cadherin-DCs,炎性E-cadherin+DC能促使初始(naive) CD8+T細(xì)胞產(chǎn)生更高濃度IFN-γ.5. E-cadherin+DC體內(nèi)對CD4+T細(xì)胞亞群分化的作用類似體外,同時刺激了大量CEA特異性效應(yīng)CD8+T細(xì)胞(第14天,E-ca+組VS E-cad-組2.01%VS1.31%,第21天,E-cad+組VS E-cad-組3.47%VS1.94%,第28天,E-cad+組VS E-cad-組4.85%VS2.57%)及CD103+CD8+T細(xì)胞(E-cad+組VS E-cad-組15.20%VS3.70%)的產(chǎn)生,發(fā)揮了良好的抗腫瘤效應(yīng)。 結(jié)論：anti-CD40誘導(dǎo)的E-cadherin+DCs能增強lewis肺癌模型抗腫瘤T細(xì)胞免疫反應(yīng)并表現(xiàn)出良好的抗腫瘤能力。這為未來anti-CD40單克隆抗體應(yīng)用于肺癌免疫治療及肺癌樹突狀細(xì)胞疫苗治療開辟了新的思路。
[Abstract]:Research background and objective: lung cancer is one of the most common malignant tumors in China. According to the cancer registration center, in 2010, 605 thousand and 900 new cases of lung cancer in China were the first malignant tumor, and the number of lung cancer deaths in China was 486 thousand and 600 in the same period, accounting for the cause of the death of malignant tumors, the main treatment methods for lung cancer were surgery, radiotherapy, chemotherapy and 24.87%.. However, there is no traditional therapy for the treatment of lung cancer. With the continuous development and renewal of the treatment, the immunotherapy and the pathogenesis of the immune microenvironment of lung cancer have entered a new era. This provides a new way of treatment for the individualized treatment of lung cancer and a thorough cure for the lung. Cancer has brought hope. Because of lung cancer, it is different from malignant melanoma and has no immunogenicity. It is not easy to be found and bound by T cells in the tumor microenvironment. At the same time, it can promote the regulation of the differentiation of T cells (Treg) and the secretion of corresponding cytokines, so as to inhibit the reaction of their own anti-tumor T cells. The main way to enhance the anti-tumor T cell response is to block the checkpoint of inhibition of T cell activation pathway with monoclonal antibodies (such as anti-PD-1 and anti-CTLA-4.).
But recently co stimulation of monoclonal antibody anti-CD40 provides a new way of immunotherapy for lung cancer immunotherapy, and it has shown good antitumor effects in a phase study of pancreatic cancer and diffuse large B cell lymphoma. But anti-CD40 stimulates the body with cytokine release syndrome, thrombus and other adverse reactions. It is known that Anti-CD40 has the dual role of activating the antigen presenting cell (APC) to induce the inflammatory reaction and the good antitumor effect in the tumor microenvironment. In order to make up for the deficiency of anti-CD40, the study of the mechanism of the anti-tumor mechanism of inflammatory dendritic cells in the anti-CD40 mediated inflammatory reaction will be anti-CD 40 monoclonal antibodies are better used in the field of immunotherapy for lung cancer dendritic cells. At the same time, the immunotherapy of lung cancer based on dendritic cells (DCs) has become a new research hotspot. However, the biological functions and regulatory mechanism of different DCs subgroups are not clear, which will lead to the clinical application and treatment. The new study found that in the Anti-CD40 induced inflammatory response, a new type of inflammatory dendritic cell subtype E-cadherin positive dendritic cells were found by Professor Fiona Powrie. But the role and mechanism of this kind of dendritic cells in the immune microenvironment of lung cancer is unknown. This experiment is mainly to explore the new type of inflammation. The role and mechanism of E-cadherin positive dendritic cells in the anti-tumor T cell immunity of Lewis cancer models.
Materials and methods: 1. using FGK45 cell supernatant to prepare mouse anti-CD40 antibody, and then use rag1KO mice and anti-CD40 antibody to prepare anti-CD40 inflammation animal model.2. to take Lewis lung cancer model, C57/BL6 mouse, Rag1KO mouse and anti-CD40 inflammation animal model mice lung tissue, extract the 4 kinds of immune state of lung tissue and its single The distribution of E-cadherin positive dendritic cells (DC) and phenotypic.3. were used to detect the distribution of DC and the phenotypic.3. to extract the single cells of the spleen from the animal model of the inflammatory animal. The flow formula was used to isolate the E-cadherin+DC, and the CEA421-435 antigen peptides were co cultured with the initial (naive) CD4+T cells, and the intracellular and cellular cells were used. The analysis of anti-CD40 induced inflammatory E-cadherin+DC induced the initial CD4+T cell effect (effector) T cells (Thl, Th2 and Th17) and the differentiation of T cells (Treg) in the tumor microenvironment. The stimulation effect CD8+T cells produce IFN- gamma level.5., and anti-CD40 induced E-cadherin+DCs loaded CEA526-533 antigen peptide is injected into the situ model of Lewis lung cancer through the tail vein to detect the dynamic changes of CEA tetramer+ (four polymer) CD8+T cells in the body and the change of CD103+CD8+T cells in the body. At the same time, the microenvironment CD is detected by lung cancer and lung tissue on the twenty-eighth day. Changes in 4+T cell subsets.
Results: 1. the successful preparation of high purity anti-CD40 antibody and anti-CD40 inflammation animal model.2.E-cadherin+DC mainly concentrated in the lungs of the animal model of anti-CD40 inflammation, while in the Lewis City cancer model, no obvious aggregation was found in the lungs of normal C57/BL6 mice and Rag1KO mice. The inflammatory E-cadherin+DC induced by the lung anti-CD40 was similar to the spleen inflammatory E-ca. The dherin+DC phenotype, which is the E-cadherin+CD11chighCD11b+CD103-CD4-.3. E-cadherin+DC carrying CEA antigen peptide in vitro, promotes the differentiation of initial CD4+T cells to Thl, Th17 cells, and inhibits the differentiation of Th2 and Treg cells,.4. is higher than E-cadherin-DCs. The effect of +DC on the differentiation of CD4+T cell subsets was similar in vitro, and a large number of CEA specific effect CD8+T cells were stimulated at the same time (Fourteenth days, 2.01%VS1.31% of group VS E-cad- in group E-ca+, twenty-first days, VS E-cad- group 3.47%VS1.94% of E-cad+ group, twenty-eighth days. A good antitumor effect.
Conclusion: anti-CD40 induced E-cadherin+DCs can enhance the anti tumor T cell immune response of Lewis lung cancer model and exhibit good anti-tumor ability, which opens up a new idea for the application of anti-CD40 monoclonal antibody in the treatment of lung cancer immunotherapy and the treatment of lung cancer dendritic cell vaccine.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R734.2

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